Enthalpy Changes during Photosynthetic Water Oxidation Tracked by Time-Resolved Calorimetry Using a Photothermal Beam Deflection Technique  Roland Krivanek, Holger Dau, Michael Haumann  Biophysical Journal  Volume 94, Issue 5, Pages 1890-1903 (March 2008) DOI: 10.1529/biophysj.107.117085 Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 1 Extended reaction scheme of the S-state cycle of photosynthetic water oxidation at the Mn complex of PSII (9–12). The cycle comprises eight steps of alternate electron and proton removal from the Mn complex. The S3n state corresponds to the S4 state described in (9); S4+ is a hypothetical intermediate where four protons and four electrons have been abstracted from the Mn complex before O2 release. Within <1μs after each flash, YZ⋅+ is formed. The halftimes of the transitions between the semistable S states (bold characters) were taken from (9). The superscripts “n” (neutral) and “+” (one positive charge) denote the overall charge of the Mn complex resulting from the sum of electron abstraction (+1/e−) and proton release (−1/H+) up to the respective S state. For a detailed discussion of the reaction cycle, see (9–12). Biophysical Journal 2008 94, 1890-1903DOI: (10.1529/biophysj.107.117085) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 2 Photothermal beam deflection (PBD) experiment. (A) Schematic drawing of the experimental setup in our laboratory. Lenses for beam shaping are omitted for clarity. Excitation of the sample by a pump-laser flash (532nm) and subsequent heat release and nonthermal changes in the sample lead to the deflection of the continuous-wave probe beam (920nm) by an angle φ, resulting in the PBD signal. (B) Normalized intensity profiles and relative positioning of pump- and probe-laser beams as measured by a photodiode in the center of the sample cuvette. The FWHM values are 0.26mm(920nm) and 0.54mm(532nm). (C) PBD signal of the calorimetric standard BCP; 1000 transients have been averaged. (Upper inset) The signal rise time of ∼3μs is limited by the differential-photodiode detector. (Lower inset) Within 20ms after the exciting flash, the signal decays by only ∼3% of its initial amplitude. Biophysical Journal 2008 94, 1890-1903DOI: (10.1529/biophysj.107.117085) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 3 Absorption spectra of a PSII membrane sample, the dye BCP, a mixture of PSII and BCP, and the buffer. That the difference spectrum (PSII+BCP)-BCP is identical to that of PSII alone indicates that the spectrum of PSII was unchanged in the presence of BCP, and vice versa. The pump- and probe-laser wavelengths are indicated by arrows. From the spectra of BCP and PSII, the background absorption of the buffer was subtracted. Spectra of solutions containing PSII additionally were corrected for scattering contributions. From the spectra of PSII and BCP, the absorption (Aexc) at the excitation wavelength (532nm) was determined. Biophysical Journal 2008 94, 1890-1903DOI: (10.1529/biophysj.107.117085) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 4 PBD signals of PSII membranes for repetitive pump-flash excitation. About 1000 signals from five samples were averaged; the time resolution was 5μs/data point (Eexc=6μJ, T=20°C). A standard PSII sample (20μg Chl/mL, 20μM DCBQ) was used as the control (upper trace). The slow phase (Xms) was simulated (line) using an exponential function with t1/2=1.15ms. (Middle trace) Sample with 40μMDCMU+20μMDCBQ. The small and rapid signal decay after the exciting flash may reflect a contribution due to the S1n→S2+ transition. (Lower trace) Sample with 40μMDCMU and 20μMDCBQ, with additional continuous illumination. Biophysical Journal 2008 94, 1890-1903DOI: (10.1529/biophysj.107.117085) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 5 (A) PBD signals induced by the exciting pump flash (arrows) after the application, preceding the pump flash, of zero to seven saturating flashes to initially dark-adapted PSII membranes. Eexc=8.5μJ, ∼200 transients were averaged, the time resolution was 50μs/data point, and T=20°C. The rising signals in the millisecond time range (Xms) were simulated using a t1/2 of 1.15ms, as exemplified for the transient on the third flash (line). (B) The amplitude of Xms (circles) as a function of the total flash number (saturating flashes plus pump flash). The solid line represents a simulation using the Kok model (69) and 10% of misses and 100% of centers in state S1n before light-flash application. Biophysical Journal 2008 94, 1890-1903DOI: (10.1529/biophysj.107.117085) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 6 PBD signals of PSII membranes at three pump-flash energies (at 20°C). The slow phase (Xms) of the three signals was simulated using an exponential function with t1/2=1.15ms. Note the increase of Xprompt (dashes) relative to Xms at increasing Eexc. Biophysical Journal 2008 94, 1890-1903DOI: (10.1529/biophysj.107.117085) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 7 Dependence of the PBD signals of PSII on the energy (Eexc) of the pump flash. (A) Relative magnitudes of Xms (solid circles) reveal single-exponential saturation behavior (line). (B) Relative magnitudes of PBD signals (Xprompt/Eexc) due to instantaneous heat release. Xprompts are from data in Figs. 4 (+DCMU) (open circles) and 6 (solid circles). Data points were simulated (line) using Eq. 9 (Eexc1/2=4.0±0.6μJ,⁡xmax=5.95⁡μJ). Solid triangles represent Xprompt from data in Fig. 4 (+DCMU+light). The dashed line accounts for the conversion of 100% of the absorbed excitation energy into heat. Biophysical Journal 2008 94, 1890-1903DOI: (10.1529/biophysj.107.117085) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 8 (A) PBD signals of PSII membranes for repetitive flash excitation in the absence (upper trace) and presence (middle trace) of BCP (6.8μg/mL, similar absorption of 0.14 at 532nm to that of PSII membranes); the lower trace represents the difference signal. About 1000 transients were averaged; the time resolution was 10μs/data point (Eexc=5μJ, T=20°C). A signal of BCP in buffer (triangles) scaled to the magnitude of that resulting from the measurement in the presence of PSII is shown for comparison (lower trace). (B) Xprompt from measurements similar to those in A of BCP and PSII membranes at increasing absorption at 532nm determined from spectra shown in Fig. 3. The dashed line denotes the absorption (Aexc) of 0.14 of the PSII membranes at a Chl concentration of 20μg/mL. Biophysical Journal 2008 94, 1890-1903DOI: (10.1529/biophysj.107.117085) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 9 PBD signals of PSII at the indicated excitation energies and buffer temperatures. About 1000 transients at 10μs/data point (5μJ) and 5000 transients at 20μs/data point (1.2μJ) were averaged. Xms (dashed lines) was simulated (solid line) using t1/2 values of 0.77ms (32.5°C), 1.45ms (12.5°C), and 2.20ms (−0.1°C) at Eexc=5μJ, and of 0.79ms (30°C) and 2.27ms (−1°C), respectively, at Eexc=1.2μJ. Note that the magnitude of Xms is similar in all traces, whereas Xprompt strongly decreases with decreasing temperature. Biophysical Journal 2008 94, 1890-1903DOI: (10.1529/biophysj.107.117085) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 10 Arrhenius plot of the rate constant of the millisecond phase (Xms) of PBD signals due to the oxygen-evolving transition. Data points were derived from measurements at Eexc=5μJ similar to those in Fig. 9left, and slightly smoothed by adjacent averaging over two points for display. The line represents a fit using the Arrhenius equation and an activation energy (Ea) of 230meV (k=k0(exp(−Ea/kBT)); kB is the Boltzmann constant, and the value of the preexponential factor, k0, was 5.75×103 ms−1). Biophysical Journal 2008 94, 1890-1903DOI: (10.1529/biophysj.107.117085) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 11 Temperature dependence of Xprompt (left) and Xms (right) of PSII (solid circles) compared to the PBD signal of the standard BCP (triangles) at three excitation energies (for equal absorption at 532nm). Bold lines represent fit curves calculated using Eqs. 4 (BCP) and 5 (PSII) and the parameters shown in Table 1. PBD signals of BCP were measured in the presence (open triangles) or absence (solid triangles) of PSII membranes. At T0=−16±1°C (dotted lines), the thermal contributions to the PBD signal vanish and only the ΔN-related signals remain. ΔN is negative for YZ⋅+QA− formation (Xprompt), but positive for the O2-evolving transition (Xms). From the fit curves, the magnitudes of ΔN, QSt, and ΔQ (arrows) in Table 1 were determined. In the middle chart in the left column, the amplitudes of PBD signals of BCP measured in pure water (+) and a respective simulation yielding T00=0∘C (thin line) are shown for comparison. Biophysical Journal 2008 94, 1890-1903DOI: (10.1529/biophysj.107.117085) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 12 Fractions of the absorbed energy of a 532-nm photon that are released as heat (ΔQ) from PSII upon YZ⋅+QA− formation (solid circles, left y axis) and upon the O2-evolving transition (open squares, right y axis) plotted versus the excitation energy. Data points correspond to the values listed in Table 1. Curves represent fits to the data (solid line, ΔQprompt; dashed line, ΔQms) using a saturation behavior as described by Eq. 9 and a value of Eexc1/2 of 4μJ (derived from Xprompt (see Fig. 7 B)). Dotted lines denote the ΔQ values from extrapolations of the simulation curves to Eexc=0. Biophysical Journal 2008 94, 1890-1903DOI: (10.1529/biophysj.107.117085) Copyright © 2008 The Biophysical Society Terms and Conditions